Focal Adhesion Kinase (FAK) is a critical survival signal in cancer and a promising therapeutic target that is being evaluated in several clinical trials using kinase enzyme inhibitors. The FAK scaffold provides a new area for developing highly specific therapeutics against its interactome and can be personalized to an individual tumor phenotype. During this period of support, we have demonstrated that we can successfully target the FAK scaffold with small molecule inhibitors, block FAK Y397 autophosphorylation, inhibit FAK- dependent downstream signaling, and develop effective therapeutic approaches. We hypothesize that there are distinct FAK-driven cellular scenarios which require the need for FAK scaffold and/or kinase inhibitors in order to achieve an optimal tumor response. We also hypothesize there are distinct tumor phenotypes that will cause resistance to FAK inhibitors, and by identifying these molecular mechanisms we will develop novel therapeutic approaches to overcome resistance. The first specific aim will define the molecular mechanisms of sensitivity to FAK scaffold inhibitors compared to FAK kinase inhibitors. We will determine the direct effects of FAK scaffolding partners HER-2 and VEGFR-3 on FAK trans- phosphorylation and downstream signaling in the absence of FAK kinase activity. We will analyze our prototype FAK scaffold inhibitors for blocking FAK trans-phosphorylation and downstream cellular phenotypes. We will characterize our novel FAK scaffold inhibitors that target both the Y397 autophosphorylation site and FAK-HER-2 binding, using molecular, biochemical, cellular and structural readouts. The second specific aim will define the mechanism of resistance to FAK-Y397 scaffold inhibitors. We will test the mechanism of resistance and sensitivity to FAK-Y397 scaffold and FAK kinase inhibitors in cancer stem cells (CSCs) and patient-derived xenograft (PDX) CSC models. We will test the mechanism of resistance and sensitivity to FAK Y397 inhibitors by using a combination therapy approach with inhibitors of cancer stem cell markers and targeted therapeutics. We will determine new approaches to overcome resistance to FAK Y397 inhibitors by using a combinational therapy approach with inhibitors of cancer stem cell markers and targeted therapeutics. We will test synergy of FAK-Y397 scaffold, FAK kinase, and other FAK-scaffold inhibitors to overcome resistance. The third specific aim will develop FAK-related biomarkers in human tumor samples that predict response to FAK-targeted therapeutics. We will correlate tumor expression of FAK (total and phosphorylated), selected RTKs, CSC-markers, and other proposed FAK biomarkers with patient and tumor-specific data and outcomes using tissue microarrays (TMAs) and our extensive Roswell Park Cancer Institute (RPCI) databases and biorepository. Using data from these aims, we will evaluate tumor response to mono- and combinational FAK-therapy (scaffold and kinase) in FAKlow and FAKhigh stratified breast PDX models to validate our biomarker studies.